Structure and function of DnaB helicase and its eukaryotic homologs

DNA helicases play important roles in pathways of nucleic acid metabolism. The structure and function of E. coli DnaB helicase and two homologs from yeast and human have been analyzed, and found similar to DnaB helicase. Three distinct structural domains of the 471 residue DnaB helicase were defined: domain $a$amino acid (aa) 1–156, domain $b$ aa 157–302 and domain $g$ 302–471. Deletions in these domains were examined for hexamer formation, DNA-dependent ATPase, and DNA helicase activities. These studies showed: (i) domain $b$ contained a functional ATPase active site; (ii) domain $g$ appeared to be the DNA binding domain and modulator of ATPase activity; (iii) domain $a$ plays a pivotal role in transducing the energy of ATP hydrolysis to DNA unwinding and (iv) all three domains are required for helicase activity. Further deletion, and in vitro mutagenesis studies of domain $g$ demonstrated that a basic region, “RSRARR”, and leucine zipper motif are required for ssDNA binding.; An analogous DNA helicase, previously identified as a component of the multi-protein polymerase a complex from the yeast S. cerevisiae, has been purified to homogeneity. HCS1, the gene for yeast helicase A (yHcsA) was identified and the protein was expressed in E. coli. Yeast HcsA shares certain features with other hexameric DNA helicases: (i) it is hexameric with monomer-hexamer dynamics similar to SV40 T-antigen; (ii) it interacts with pol $a$-primase complex; (iii) its helicase activity is stimulated by ssDNA binding proteins; and (iv) it has 5′–3 ′ polarity of movement.; Sequence alignment of the primary structure of yHcs A demonstrated that it is homologous to DNA helicases from human (50%) and C. elegans (40%) as well as DnaB helicase (25%). Analysis of recombinant human helicase A (hHcsA) demonstrate several similarities between these helicases: (i) a strictly DNA dependent ATPase activity, (ii) DNA helicase activity, with a 5′–3′ polarity, and (iii) hexameric oligomeric structure. Analysis of the mRNA levels of this protein suggested that they fluctuate through out the cell cycle and that maximum amounts are present in G1.